The Ras superfamily of small GTPases is comprised of five major groups: Ras, Rho, Rab, Arf and Ran that regulate many aspects of cell behavior. The Ras (e.g. H-Ras, K-Ras, R-Ras and Rap1) and Rho subfamily (e.g. Cdc42, Rac1 and RhoA) of GTPases synergistically regulate signaling pathways that originate from extracellular stimuli, to yield overlapping sets of cellular phenotypes, such as proliferation, differentiation, and remodeling of the cytoskeleton. The GTPases function by cycling between active GTP-bound and inactive GDP-bound states. Guanine nucleotide-exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide-dissociation inhibitors (GDIs) (Guilluy et al., 2011; Jaffe and Hall, 2005) control the activity of the GTPases. GEFs activate Rho proteins by catalyzing the exchange of GDP for GTP, while GAPs inactivate the proteins by stimulating intrinsic GTPase activity. GDI inhibits the activation of Rho GTPases by sequestering them in the cytosol away from membranes. Activated GTPases interact with specific downstream-effector proteins to yield definite physiological responses in response to the upstream stimuli. Ras and Rho family GTPases function as components of a broader signaling network and are interconnected across overlapping signaling pathways that involve positive and negative feedback loops.
The superfamily of GTPases has numerous cellular effects that are dysregulated in disease. Ras (35 members) primarily involved in signaling and cancer. Rho (23 members) GTPases are primarily involved in cell motility, infection and cancer among others, Rab (70 members) GTPases are primarily involved in intracellular transport, cancer, infections disease, genetic disease and downstream growth factor signaling Ran (1 member) nuclear import, cellular differentiation, Arf (30 members) intracellular transport, infectious disease, human ciliopathies and retinopathies. The dysregulation of these systems can be measured as an increase in the enrichment of active GTPases caused by factors in patient samples. Active GTPases preferentially bind to specific cognate effector molecules that are immobilized on beads, thus providing evidence as to the dysregulation of the systems involved.
The interactions of viruses and host cells is known to elicit the activation of multiple GTPases to promote the cytoskeletal remodeling required for breaching inter- and intracellular cellular barriers to infection as well as intracellular trafficking of internalized virions to allow replication. Most studies investigating the role of GTPases in viral interactions with host cells use traditional methods of active GTPase pull-down and detection by Western blot, which are slow, labor intensive and require large amounts of starting material. Newer, commercially available plate-based effector binding assays for detecting activated GTPases known as GLISA (Cytoskeleton, Inc.) require less material than western blot based assays, yet are still labor intensive; requiring freezing of aliquots, protein assays to ensure linearity and numerous binding and washing steps. Accordingly, most studies tend to focus on a limited subset of GTPases, which presents significant limitations when one wants to examine the broader spectrum of cell signaling space impinged upon by viral activity. Based on these considerations, we have developed a rapid, and quantitative flow cytometry-compatible, bead-based effector binding assay to analyze, in parallel, multiple GTPases that are activated in a single virus-infected cell sample.
Sepsis is a disease that now affects more than 900,000 patients with an estimated mortality rate of 30% in the US.22-25A Annual costs are estimated to exceed $20 billion.26A Severe trauma patients who survive the initial injury are at risk of developing sepsis syndrome and multiple organ failure.24A, 25A, 27A Systemic microvascular leakage, most likely due to the release of inflammatory, coagulation and fibrinolysis factors, is a signature of sepsis in trauma patients.22A An improved understanding of the clinical mechanisms of sepsis, including the roles of pathogens, sites of injury and patient heterogeneity, is urgently needed to enable better prevention, diagnosis, and treatment.22A The goal of this project is to address the need for timely and accurate differential diagnosis of sepsis and SIRS due to sterile inflammation. The pathophysiology of sepsis involves nearly all cell types, tissues, and organ systems, and has so far been associated with about 180 distinct potential biological markers.4-8A These markers are organized as follows: vasoactive amines, vasoactive peptides, fragments of complement components, lipid mediators, cytokines, chemokines, and proteolytic enzymes involved in the coagulation and fibrinolytic system.9A This level of heterogeneity continues to confound efforts to discover universally applicable models.
A heterogeneous patient population and a diverse ensemble of pathogenic bacteria highlight a cardinal problem in defining the pathogenesis of sepsis.1-3A So far, about 180 potential biological markers of sepsis are known.4-8A Their broad-spectrum applicability to sepsis and other pathologies has limited their early diagnostic utility.9A New approaches accounting for the complexity of the inflammatory response and changes that occur during the course of sepsis are needed.
Small GTPases and their regulators, as they actuate and fine-tune pivotal molecular pathways, constitute vulnerable nodes of the cell. Their activities are associated with a diverse range of biological functionality in health and disease, such as cancer[1-3], cardiovascular diseases or developmental diseases [6], and infections [4,5]. In disease, GTPase signaling pathways are diverted during the onset or progression of the disease, and disorders in which the expression, regulation, or function of regulators is directly impaired by mutations. These include congenital diseases in which GTPase regulators carry missense mutations that impair their biochemical properties, and infections in which pathogens have created new regulators of that own to take command of host pathways. For some of these diseases, understanding the biochemical basis may help in discovering pharmaceuticals to correct these defects. Through evolution, bacterial pathogens have evolved a battery of toxins and virulence factors that target small GTPases that attenuate GTPases functions, in order to faciliatate host entry and dissemination.
1. Vega, F. M.; Ridley, A. J. Rho gtpases in cancer cell biology. FEBS letters 2008, 582, 2093-2101.
2. Iden, S.; Collard, J. G. Crosstalk between small gtpases and polarity proteins in cell polarization, Nature reviews. Molecular cell biology 2008, 9, 846-859.
3. Agola, J.; Jim, P.; Ward, H.; Basuray, S.; Wandinger-Ness, A. Rab gtpases as regulators of endocytosis, targets of disease and therapeutic opportunities. Clinical genetics 2012.
4. Lemichez, E.; Aktories, K. Hijacking of rho gtpases during bacterial infection Experimental cell research 2013, 319, 2329-2336.
5. Aktories, K.; Schmidt, G. A new turn in rho gtpase activation by escherichia coli cytotoxic necrotizing factors. Trends in microbiology 2003, 11, 152-155.
6. Cherfils, J.; Zeghouf, M. Regulation of small gtpases by gels, gaps, and gdis. Physiological reviews 2013, 93, 269-309.